Tree shrew Alzheimer's disease modeling method

Abstract

The invention discloses a tree shrew Alzheimer's disease modeling method. The method comprises the following specific steps: mixing an ethanol solution of Microcystin-LR with the concentration of 0.5 [mu] g / [mu] l with an amaninic acid solution, and injecting 2 [mu] l to Meynert basal nuclei on the two sides of a tree shrew respectively; and after drug administration is finished, normally feeding for 2 weeks to finish Alzheimer's disease modeling. The method comprises the following steps: under a sterile condition, sucking 200 microliters of sterile normal saline, adding the sterile normal saline into a small bottle containing 1mg of amanitine, repeatedly blowing and beating to completely dissolve the amanitine, and preparing a solution with the concentration of 5mu g / microliter; the Microcystin-LR solution is a product purchased in the market. According to the invention, a mixed solution of MC-LR (0.1 [mu] g) and IBO solution (3 [mu] g) is respectively injected to Meynert basal nuclei on both sides of the tree shrew, and a stable tree shrew AD model can be established after 2 weeks. After the medicine is administrated for 3 days, tree shrews have AD behavior expressions, such as drooping spirit and reduced activity; after two weeks, the tree shrew brain shows AD typical pathological expressions such as A beta deposition, increase of expression levels of multiple phosphorylation sites of Tau protein and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of disease animal model construction, and in particular relates to a tree shrew Alzheimer's disease modeling method. Background Art

[0002] Alzheimer's Disease (AD), also known as senile dementia, is a common multiple, progressive neurodegenerative disease. Its main clinical manifestations are progressive memory loss, cognitive dysfunction, abnormal behavior and social disorders, and even loss of ability to live. Patients often die from complications within 10-20 years of onset. The prevalence rate shows a significant increasing trend with age. This not only puts great pressure on patients and their families, but also imposes a huge economic burden on society. Therefore, it is very necessary and urgent to seek ways to prevent AD, find new treatment approaches for AD, and develop new drugs to treat AD.

[0003] AD has an insidious course and presents progressive development. Generally, before clinical manifestations are detected, a variety of subtle pathophysiological changes have occurred in the patient's brain. These pathophysiological changes include the abnormal accumulation of Aβ toxic proteins produced by APP shearing to form amyloid plaques, a large number of neurofibrillary tangles caused by excessive phosphorylation of tubulin tau, reduced brain glucose metabolism, uncontrolled activation of microglia, chronic oxidative stress damage, and the secretion of neurotoxins and inflammatory factors. However, the specific pathogenesis has not been clearly studied so far. There are mainly β-amyloid protein cascade theory, tau protein theory, cholinergic damage theory, oxidative stress / mitochondrial cascade theory, excitatory glutamate toxicity theory and gene mutation theory. Because the pathogenesis of AD is very complex and the pathogenesis involves many links, there is currently a lack of therapeutic drugs with satisfactory efficacy. In order to explore the pathogenic mechanism and potential therapeutic drugs of AD, establishing a suitable animal model is a key step. The existing AD animal models include aging type, injection-induced type, transgenic type, and composite type. These four models are widely used in the study of AD pathogenesis and drug treatment, and each has its own advantages and disadvantages. However, the existing AD animal models take a long time to create and are inefficient, which seriously affects the progress of subsequent research. For example, it takes more than 2 months to create a D-galactose-compounded IBO model. Therefore, it is of great significance to develop an animal model that is closer to humans, takes less time, and can effectively simulate AD pathology. Summary of the invention

[0004] The purpose of the present invention is to provide a method for modeling Alzheimer's disease in tree shrews.

[0005] The object of the present invention is achieved by providing a tree shrew Alzheimer's disease modeling method, the specific steps of which are as follows: (1) Reagent preparation: Microcystin-LR solution: an ethanol solution of Microcystin-LR at a concentration of 0.5 μg / μl; Microcystin-LR is a highly effective protein phosphatase inhibitor, referred to as MC-LR, which is microcystin or microcystin, and is a commercially available product purchased from MCE Company; Preparation of ibotenic acid solution: Under sterile conditions, pipette 200 μl of sterile saline into a vial containing 1 mg of ibotenic acid, and repeatedly pipette to completely dissolve it. The concentration is 5 μg / μl. (2) Mix the Microcystin-LR solution prepared in step (1) with the ibotenic acid solution and inject 2 μl of the solution into the bilateral Meynert nucleus of the tree shrew. The mixed solution is obtained by diluting the solution under sterile conditions in the ratio of Microcystin-LR: ibotenic acid solution: sterile saline at 1:3:6. (3) After the administration, the mice were kept normally for 2 weeks to complete the Alzheimer's disease model.

[0006] The beneficial effects of the present invention are: The present invention injects a mixed solution of MC-LR (0.1 μg) + IBO solution (3 μg) into the bilateral Meynert basal nuclei of tree shrews, and a stable tree shrew AD model can be established after 2 weeks. Three days after administration, tree shrews show AD behavioral manifestations, such as mental depression and reduced activity; two weeks later, the tree shrew brain shows typical AD pathological manifestations such as Aβ deposition and increased expression levels of multiple phosphorylation sites of Tau protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 This is a comparison chart of the Aβ protein expression in the hippocampus and cortex of tree shrews in the five comparative experiments of this application; Figure 2 This is a comparison chart of the Aβ protein expression in the hippocampus and cortex of tree shrews in the five comparative experiments of this application; Figure 3 This is a comparison chart of the expression changes of p-Tau (T231), p-Tau (S396), and Tau proteins in tree shrews in the five comparative experiments of this application; Figure 4 This is a comparison chart of the changes in p-Tau (T231) protein expression in tree shrews in the five comparative experiments of this application; Figure 5 This is a comparison chart of the expression changes of p-Tau (S396) in tree shrews in the five comparative experiments of this application; Figure 6 This is a comparison chart of the changes in Tau protein expression in tree shrews in the five comparative experiments of this application. DETAILED DESCRIPTION

[0008] The technical solution of the present invention will be described clearly and completely in combination with the embodiments below. Obviously, the described embodiments are only part of the embodiments of the present invention, but not all of them. All other embodiments belong to the protection scope of the present invention.

[0009] 1. Experimental Animals Thirty ordinary male Burmese tree shrews weighing 120-150 g were purchased from the Institute of Medical Biology, Chinese Academy of Medical Sciences [SCXK (Yunnan) K2023-0003] and kept in the ordinary animal room of Yunnan Luoyu Biotechnology Co., Ltd. [SYXK (Yunnan) K2021-0003] in stainless steel cages at room temperature (24±2)℃, relative humidity (50±5)%, and light / dark cycle of 12 h / 12 ​​h.

[0010] 2. Main reagents and consumables Reagent name company Microcystin-LR Mce Ibotenic acid Mce β-Amyloid Antibodies Abcam Tau Antibody Abcam p-Tau (T231) Antibody Abcam p-Tau (S396) Antibody Abcam GAPDH Antibody Abcam Anti-rabbit IgG CST RIPA Lysis Buffer (Strong) Blue Sky Prestained protein marker Thermo Fisher Bovine serum albumin (BSA) Sigma SurperECLPlus Ultrasensitive Luminescence Prile Sheep serum for blocking Zhongshan Jinqiao Universal two-step test kit Zhongshan Jinqiao DAB colorimetric kit Zhongshan Jinqiao PVDF membrane Millipore 3. Main instruments Instrument Name company Brain Stereotaxic Instrument Raymond Micro-injection pump Lange Nanophotometer ultra-micro-volume spectrophotometer IMPLEN Protein electrophoresis tank, electrotransfer tank, power supply Bio-Rad ECL Luminescence Imager Tanon Automatic tissue dehydrator Wuhan Junjie Paraffin embedding machine German Leica Rotary microtome German Leica microscope OLYMPUS BX53 4. Preparation of reagents Microcystin-LR (MC-LR): The purchased reagent is an ethanol solution of MC-LR at a concentration of 0.5 μg / μl.

[0011] Ibotenic acid (IBO): Under sterile conditions, pipette 200 μl of sterile saline into a vial containing 1 mg of IBO and pipette repeatedly to dissolve it completely to a concentration of 5 mg / ml (5 μg / μl).

[0012] 5. Experimental methods 5.1 Animal model preparation Tree shrews were randomly divided into the following five groups ①, Normal (NC) group: Tree shrews were raised normally without any treatment ②Sham group: 2 μl of saline (containing 20% ​​ethanol) was injected into bilateral Meynert nucleus basalis ③, MC-LR group: 2 μl of 0.05 μg / μl MC-LR solution was injected into each of the bilateral Meynert basal nuclei (under sterile conditions, 0.5 μg / μl MC-LR: sterile saline = 1:9 dilution) ④, IBO group: 2 μl of 1.5 μg / μl IBO solution was injected into each of the bilateral Meynert basal nuclei (under sterile conditions, 5 μg / μl IBO solution: sterile saline = 3:7 for dilution); ⑤. MC-LR+IBO combined group: 2 μl of a mixed solution of MC-LR (0.05 μg / μl) + IBO solution (1.5 μg / μl) was injected into each of the bilateral Meynert basal nuclei (under sterile conditions, 0.5 μg / μl MC-LR: 5 μg / μl IBO solution: sterile saline = 1:3:6 dilution) 5.2 Bilateral Meynert basal nucleus (BM) injection method (1) Fixing the tree shrew: The tree shrew was anesthetized by intraperitoneal injection of 3% sodium pentobarbital at a rate of 2 μl / g body weight. After complete anesthesia, the hair on the tree shrew's head and face was removed with animal clippers, and the tree shrew was fixed on a stereotaxic apparatus: the ear rod was inserted into the ear canal of the tree shrew, and the left and right ear rods were balanced and adjusted so that the line between the two ears of the tree shrew and the ear rods were in the same straight line; the incisors of the tree shrew were opened with tweezers, and the upper incisors of the tree shrew were stuck in the hole of the adapter, and the nose rod locking screw was pressed down. At the same time, the height of the incisor clamp was adjusted up and down, and the position of the adapter was adjusted forward and backward to keep the cranial face of the tree shrew level, and the screw was locked to prevent the tree shrew's head from shaking; according to the body size of the tree shrew, the body of the tree shrew was raised with foam on the base of the locator so that the head and body were level to prevent the tree shrew from breathing obstruction.

[0013] (2) Microinjection pump parameter setting: Turn on the power switch of the microinjection pump, set the operation mode to extraction first and then infusion, set the extraction parameters: the extraction volume is 2μl, the extraction time is 0.5min; the interval time between extraction and infusion is 0.5min; set the infusion time to 5min.

[0014] (3) Locating the reference point: After fixing the RN type injection needle (specification 10 μL) on the clamp, turn on the power switch of the digital locator, disinfect the skin surface of the tree shrew's skull with iodine, make a longitudinal incision from the line connecting the two ears to the eyes to expose the skull, find the intersection of the interaural line and the sagittal suture, that is, the bregma point, and use this point as the reference point of the three-dimensional coordinate system. Move the three-dimensional operating arm so that its positioning point is aligned with the bregma point, and press the "CLR" key on the digital display base X, Y, and Z axes to return to zero (desktop digital locator).

[0015] (4) Locating the target area: Determine the coordinate values ​​of the basal nucleus relative to the bregma, that is, the ML value (X axis), AP value (Y axis), and DV value (Z axis). Starting from the bregma reference point, set coordinate point 1 according to the position of the basal nucleus: AP: 8.7 mm in front of the bregma, ML: sagittal suture ±3 mm. Move the operating arm the corresponding distance (absolute values ​​of AP and ML) to the target position (Z axis does not move), then use the positioning pin as a reference point and mark it with a marker; move the "cross" operating arm and drill a hole in the skull.

[0016] (5) Experiment: After the microinjection pump is turned on to extract the prepared composite solution, the operating arm is moved downward to the target depth within 0.5 min according to the determined value DV (Z-axis coordinate of point 1): 10 mm below the dura mater. After the subsequent injection is completed, the needle is retained for 5 min, and then the needle is slowly withdrawn at a speed of 1.0 mm / min. The coordinate value is set according to the method of locating the target area to perform an injection experiment at another coordinate point. After the injection is completed, the RN injection needle is withdrawn, the injection hole is glued with bone glue, the wound is sutured and disinfected with iodine, and an appropriate amount of erythromycin ointment is applied to the wound. After surgery, intraperitoneal injection of penicillin (22000IU / kg / d) is given for 3 consecutive days.

[0017] 5.3 Behavioral observations of tree shrews After the injection, the diet, exercise, coat color and mental state of each group of tree shrews were observed daily.

[0018] 5.3 Immunohistochemical detection of Aβ expression After the administration of the drug, the tree shrews were kept normally for 2 weeks and then the tree shrews were collected. After the tree shrews were anesthetized, physiological saline and 4% paraformaldehyde solution were perfused through the heart, and the brain was taken and placed in 4% paraformaldehyde to prepare paraffin sections for subsequent immunohistochemical staining.

[0019] Immunohistochemical staining: paraffin sections were dewaxed to water, and the sections were immersed in an antigen repair box with pH=6.0 citrate buffer in a microwave oven for antigen repair. 3% hydrogen peroxide was used to block the sections in the dark for 30 minutes (to eliminate the activity of endogenous peroxidase); 5% sheep serum was used to block the sections for 1 hour, and then the primary antibody (Aβ1:400) was added and incubated overnight at 4°C. After rinsing with PBST, the universal two-step detection reagent was added and incubated at room temperature for 30 minutes; after rinsing with PBST, freshly prepared DAB color development solution was added and incubated at room temperature in the dark for 5-15 minutes. Color development was terminated with distilled water; hematoxylin counterstaining, 1% hydrochloric acid ethanol differentiation, conventional dehydration, transparency, neutral gum sealing, and the color development results were observed under an ordinary optical microscope and photographed. 5.4 Western bolt detection After the administration of the tree shrews, they were kept normally for 2 weeks and then collected. After the tree shrews were anesthetized, the chest was opened and the heart was perfused with normal saline. After the liver turned white, the tree shrew brain tissue was removed, the brain was separated on ice, and gently rinsed with 0.1mol / L PBS. 500μL of RIPA lysis buffer (containing 50μL protease inhibitor) was added for every 100mg of fresh tissue. After the tissue was fully minced with sterile ophthalmic scissors, it was homogenized with an ultrasonic cell disruptor for 20s in an ice bath environment, and then left to lyse for 20min on an ice bath. Centrifuge at 4℃, 12000rpm for 10min, collect the supernatant, aspirate about 360μL of supernatant in each tube, add 90μL 5× loading buffer (1 / 4 of the supernatant volume), mix thoroughly, centrifuge for 10s, and boil in a 100℃ metal bath for 10min to fully denature the protein. After the sample cooled, measure the protein concentration. Then, SDS-PAGE protein electrophoresis was performed, using 80V constant voltage electrophoresis until the lower edge of the sample touched the separation gel (about 30 minutes), and then changing to 130V constant voltage to continue electrophoresis for nearly 2 hours, until the bromophenol blue just overflowed the lower edge of the separation gel and stopped electrophoresis; wet transfer was performed at 280mA / 1h, and 5% skim milk was added for blocking at room temperature for 2h, and primary antibodies (rabbit anti-Tau (phospho T231) antibody 1:1000, rabbit anti-Tau (phospho S396) antibody 1:1000, rabbit anti-Tau antibody 1:1000, GAPDH antibody 1:2000) were added and incubated overnight in a silent mixer in a 4℃ refrigerator, and the membrane was washed 3 times with TBST buffer, incubated with horseradish peroxidase-labeled goat anti-rabbit secondary antibody (1:1000) at room temperature for 2h, and washed 3 times with TBST buffer. ECL reagent was added using the chemiluminescence method and developed on a gel imaging system.

[0020] 6. Experimental results 6.1 Behavioral observations of tree shrews Three days after drug injection, the tree shrews in the MC-LR+IBO combined group became listless and their activity decreased. As time went on, their activity further decreased and their hair glossiness worsened. Seven days after drug injection, the activity of the tree shrews in the IBO group decreased slightly compared with the previous few days, but there were no other abnormalities. The activities of the tree shrews in the other three groups were normal, and no abnormalities were observed.

[0021] 6.2 Immunohistochemical detection of Aβ expression In order to explore the damage caused by MC-LR and IBO to the tree shrew brain, we used immunohistochemistry to detect the expression of Aβ protein in the tree shrew brain. The results showed that compared with the NC group, the injection of MC-LR or (and) IBO increased the expression of Aβ in the hippocampus and cortex of the tree shrew brain compared with the control group, and the combined injection of MC-LR+IBO increased the expression of Aβ most significantly.

[0022] 6.3 Western bolt detection Western bolt was used to measure the protein levels of Tau, p-Tau (T231) and p-Tau (S396) in the brain tissue of tree shrews. The results showed that compared with the NC group, the p-Tau (T231) level in the brain of tree shrews after injection of MC-LR or (and) IBO was significantly increased, and the difference between the IBO group and the MC-LR+IBO group was statistically significant (p < 0.05); compared with the NC group, the p-Tau (S396) level in the brain of tree shrews after injection of MC-LR or (and) IBO was significantly increased, and the difference was statistically significant (p < 0.05); there was no significant difference in the Tau protein level (p > 0.05). Table 1 Changes in the expression of p-Tau (T231), p-Tau (S396) and Tau proteins in the five groups of tree shrews during the experiment Experimental Grouping p-Tau (T231) / GAPDH p-Tau (S396) / GAPDH Tau / GAPDH NC 0.478±0.123 0.696±0.168 0.947±0.049 sham 0.488±0.152 0.719±0.129 0.963±0.076 MC-LR 0.803±0.19 <![CDATA[0.999±0.072 a ]]> 0.956±0.113 IBO 1.183±0.287 <![CDATA[0.986±0.072 a ]]> <![CDATA[1.006±0.091 b ]]> MC-LR+IBO 1.458±0.171 <![CDATA[1.302±0.159 b,c,e ]]> <![CDATA[1.008±0.142 b,d ]]> n = 3, x±s , aP <0.05, b P <0.01, compared with the NC group; c P <0.05, d P <0.01, compared with the MC-LR group; e P <0.05, f P <0.01 compared with the IBO group.

Claims

1. A method for modeling Alzheimer's disease in tree shrews, characterized in that: The steps include: (1) Reagent preparation: Microcystin-LR solution: ethanol solution of Microcystin-LR at a concentration of 0.5 μg / μl; Preparation of ibotenic acid solution: Under sterile conditions, pipette 200 μl of sterile saline into a vial containing 1 mg of ibotenic acid, and repeatedly pipette to completely dissolve it. The concentration is 5 μg / μl. (2) Mix the Microcystin-LR solution prepared in step (1) with the ibotenic acid solution and inject 2 μl of the solution into the bilateral nucleus basalis of tree shrews. The mixed solution is obtained by diluting the solution under sterile conditions in the ratio of Microcystin-LR: ibotenic acid solution: sterile saline at a ratio of 1:3:6; (3) After the administration, the mice were kept normally for 2 weeks to complete the Alzheimer's disease model.

2. The method for modeling Alzheimer's disease in tree shrews according to claim 1, characterized in that: The specific method of bilateral Meynert basal nucleus injection is as follows: (1) Fixing the tree shrew: inject 3% sodium pentobarbital intraperitoneally at 2 μl / g body weight to anesthetize the tree shrew. After complete anesthesia, use animal clippers to remove the hair on the tree shrew's head and fix it on the brain stereotaxic instrument: insert the ear rod into the tree shrew's ear canal, balance and adjust the left and right ear rods so that the line between the two ears of the tree shrew is in the same straight line with the ear rod; use tweezers to open the tree shrew's incisors, clamp the tree shrew's upper incisors into the hole of the adapter, press down the nose rod locking screw, and at the same time adjust the incisor clamp height up and down, and adjust the adapter position forward and backward to keep the tree shrew's head and face level, and lock the screw to prevent the tree shrew's head from shaking; according to the tree shrew's body size, use foam to pad the tree shrew's body on the base of the locator so that the head and body are level to prevent the tree shrew's breathing from being blocked; (2) Microinjection pump parameter setting: Turn on the power switch of the microinjection pump, set the operation mode to extraction first and then perfusion, set the extraction parameters: the extraction volume is 2μl, the extraction time is 0.5min; the interval between extraction and perfusion is 0.5min; set the perfusion time to 5min; (3) Locating the reference point: After fixing the RN type injection needle (specification 10 μL) on the holder, turn on the power switch of the digital locator, disinfect the surface of the tree shrew's skull skin with iodine, make a longitudinal incision from the line connecting the two ears to the eyes to expose the skull, find the intersection of the interaural line and the sagittal suture, i.e., the bregma point, and use this point as the reference point of the three-dimensional coordinate system. Move the three-dimensional operating arm so that its positioning point is aligned with the bregma point, and press the "CLR" key on the digital display base X, Y, and Z axes to return to zero (desktop digital locator); (4) Locating the target area: determine the coordinate values ​​of the basal nucleus relative to the bregma, i.e., the ML value (X axis), AP value (Y axis), and DV value (Z axis). Starting from the bregma reference point, set coordinate point 1 according to the position of the basal nucleus. Method: AP: 8.7 mm in front of the bregma, ML: sagittal suture ±3 mm. Move the operating arm the corresponding distance (absolute values ​​of AP and ML) to the target position (Z axis does not move), then use the positioning pin as a reference point and mark it with a marker; move the "cross" operating arm and drill the hole; (5) Complete the injection: After the microinjection pump is turned on to extract the prepared compound solution, the operating arm is moved downward to the target depth within 0.5 min according to the determined value DV (Z-axis coordinate of point 1): 10 mm below the dura mater. After the subsequent injection is completed, the needle is retained for 5 min, and then the needle is slowly withdrawn at a speed of 1.0 mm / min. The coordinate value is set according to the method of locating the target area to perform an injection experiment at another coordinate point; After the injection, the RN injection needle was removed, the injection hole was sealed with bone glue, the wound was sutured and disinfected with iodine, and an appropriate amount of erythromycin ointment was applied to the wound. After the operation, intraperitoneal injection of penicillin (22000IU / kg / d) was given for 3 consecutive days.

3. The method for modeling Alzheimer's disease in tree shrews according to claim 1, characterized in that: The tree shrew has a body mass of 120-150 g.

4. The method for modeling Alzheimer's disease in tree shrews according to claim 1, characterized in that: The tree shrews are kept at a room temperature of 24±2°C, a relative humidity of 50±5%, and a light / dark cycle of 12h / 12h.

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